Clinical and biological significance of CD34 expression in acute leukemia

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CLINICAL AND BIOLOGICAL SIGNIFICANCE OF CD34
EXPRESSION ON ACUTE LEUKEMIC CELLS
From the beginning of its production, monoclonal
antibodies (McAbs) directed against CD34 antigen,
have been extensively explored in different hematolog-
ical malignancies. Accordingly, evidence progressively
accumulated that reactivity for CD34 was almost ex-
clusive to acute leukemia blast cells while absent in
chronic mature disorders. Since then, CD34 expres-
sion has been included in most panels of McAbs used
to characterize the immunophenotype of immature
leukemic cells as a useful marker in discriminating be-
tween mature and immature hematopoietic leukemic
cells.
CD34 reagents have also been used to explore the
potential prognostic impact of CD34 expression as
well as for its utility in identifying residual immature
blast cells for minimal residual disease monitoring.
However, the extended use of many different types of
CD34 monoclonal antibody clones and reagents has
lead to disturbing levels of variability as regards both
the incidence of CD34 expression in acute leukemias
and its prognostic relevance. In this paper we will re-
view current knowledge on the clinical and biological
significance of CD34 expression, first in acute myeloid
leukemia (AML) and afterwards in acute lymphoblastic
leukemias (ALL).
CD34 AND AML
Acute myeloid leukemias (AML) are considered to
be clonal disorders involving early hematopoietic prog-
enitor cells. A large number of recently reported pa-
pers have addressed the issue of whether it is possi-
ble to distinguish normal stem cells from progenitor
cells belonging to the leukemic clone in this disease
category. Insights into human stem cell development
together with a combined analyses of progenitor cell
phenotypic antigen expression, genetic anomalies,
and biological characteristics of acute leukemia cells
offer the perspective that distinction between benign
and malignant progenitors might be possible; these
studies may be of clinical benefit and may provide
novel tools for the treatment of this heterogeneous
group of hematological disorders (1-4). From the over-
all data it can be concluded that both differences and
similarities in the phenotypic, genotypic, and biologic
characteristics between normal and neoplastic prog-
enitors can be found in AML patients (1).
The incidence of CD34 expression on the mem-
brane of AML blasts has been found to be variable
from case to case (25-64% of the patients examined),
depending on a number of factors, many of which are
listed in Table I (5-29).
The heterogeneity in the reported incidences of
CD34
+
AML has also influenced the prognostic rele-
vance of CD34 expression in AML (4-32). In the early
nineties, a great majority of papers found a clear asso-
ciation between CD34
+
AML and both a worse progno-
sis and a lower incidence of complete remission follow-
ing induction therapy; furthermore, the relapse rate
was higher in AML showing positivity for the CD34 anti-
gen, compared to that of the CD34
-
group, thus con-
firming the negative influence of this molecule on the
clinical outcome (5-14, 20, 27, 28). However, other au-
thors could not confirm these results and found no sig-
nificant differences in the outcome and in the rate of
complete remission between CD34
+
and CD34
-
AML
patients (15-19, 21-23, 24-26, 29). Based on current
knowledge, it can be said that CD34 expression on
AML blasts does not play per se a prognostic role, but
could influence clinical outcome when associated with
some genetic lesions and/or chromosome aberrations
(24, 27, 31-34).
The heterogeneity in the reported incidence of
CD34
+
AML and its association with variable survival
rates could be explained by a number of clinical and
methodological factors (35-39). Firstly, the use of cryo-
preserved rather than fresh cells could be responsible
for a misleading interpretation of cytofluorimetric data
(37-39). In fact, freeze-thawing of AML cells can cause
Journal of Biological Regulators and Homeostatic Agents
Clinical and biological significance of CD34
expression in acute leukemia
G. BASSO
1
, F. LANZA
2
, A. ORFAO
3
, S.MORETTI
2
, G. CASTOLDI
1
Laboratory of Hemato-Oncology, Department of Pediatrics, University of Padova, Padova, Italy
2
Section of Hematology, St. Anna Hospital, University of Ferrara, Ferrara, Italy
3
Division of Citometry and Center for Cancer Research, University of Salamanca, Salamanca, Spain
J Biol Regul Homeost Agents 2001; 15: 68-78
Received:January 23, 2001
© by Wichtig Editore, 20010393-974X/068-011 $05.50

a significant increase in the number of positive cells,
due to various reasons, such as occurrence of non-
specific cross-reactivity, and vulnerability of AML cells
to freezing procedures. Staining with a nucleic acid
dye represents a pre-requisite for a reliable enumera-
tion of CD34
+
cells in cryopreserved samples (39).
A further point to be considered when evaluating
the incidence of CD34
+
AML, is represented by the
patient’s characteristics at diagnosis.The incidence of
CD34
+
cases is higher in secondary AML compared
to newly diagnosed AML. Taking into account the no-
tion that both the biology and the clinical pattern of
secondary AML significantly differ from that of de
novo AML, we can conclude that the inclusion of both
types of leukemias in a clinical study might influence
the prognostic relevance of CD34 expression in AML
patients (33, 34). It should also be considered that the
type of treatment adopted by the various authors
(chemotherapy regimen, use of allogenic and/or autol-
ogous hematopoietic stem cell transplantation) are
crucial to the clinical outcome of the disease.
Thirdly, it can be speculated that variability on the
cut-off points chosen by different authors to consider
AML blasts as carrying the CD34 molecule, may rep-
resent an additional critical factor (31,33). As the pro-
portion of CD34
+
cells is around 1% of all cells in nor-
mal bone marrow, and 0.01-0.1% in the peripheral
blood, many authors have considered 5% as the opti-
mal cut-off level for classifying an AML as being
CD34
+
. Currently, several authors agree that the cut-
off point for CD34 should be 20%, in order to avoid
misinterpretation of cytofluorimetric data. However,
there is no scientific basis for considering a sample
with 20% positive cells as positive, while another
specimen with 19% positivity as negative, since the
level of CD34 expression, as well as that of other anti-
gens, is characterized by a continuous spectrum (35,
38, 40). According to this new concept, a given sam-
ple may be regarded as positive or negative for CD34
antigen, when the mean fluorescence intensity of the
blast cell population is significantly higher than that of
normal CD34
-
mononucleated cells, or that a well-
defined population of blast cells is CD34
+
even if it
only represents a small part of all leukemic cells
present in the sample (35, 38).
Furthermore, it must be kept in mind that the choice
of a 5% cut-off level could give rise to erroneous re-
sults, since it can be influenced by the methods used
to detect antigen expression, which are characterized
by different levels of sensitivity and specificity.
Accordingly, indirect immunofluorescence stainings
used in some studies in the early nineties, are more
sensitive, although less specific, while the opposite is
true for direct immunofluorescence techniques, and
today this is the only one utilized; in fact the sensitivity
of recent commercial preparations conjugated with ef-
ficient fluorochromes is very high and produce repro-
ducible results in all laboratories (35, 38). As far as in-
strumentation is concerned, it must be underlined that
modern flow cytometers are highly sensitive in detect-
ing surface marker positivity, while microscope analy-
sis and immunoenzymatic techniques are less sensi-
tive, and produce data of lower statistical significance.
The immunophenotypic analysis of acute leukemic
blast cells should preferentially be performed on bone
marrow samples; in addition to that, even if we consid-
er a peripheral blood sample, the number of blasts p-
resent in the specimen analyzed has to be carefully e-
valuated using a multiparametric approach (32, 35-
38). However, in contrast to ALL, the discrimination of
blast cells from residual normal nucleated cells is less
likely to be obtained in AML cells by simply evaluating
the light scattering properties (forward and side scat-
ter) and the expression of CD45 antigen by the
leukemic cells. For this reason, a multiparametric ap-
proach using three-four color analysis is strongly sug-
gested in order to define the predominant leukemic
population as well as minor pathological clones or
subclones (31, 35-38). CD34 positivity should be
specifically evaluated on the blast population, in order
to avoid misinterpretation of the data. The percentage
of blasts could typically vary from 20% to 99% in the
bone marrow, and from 1 to 99% in the peripheral
blood, and therefore it is mandatory to refer the CD34
positivity to the pathological component only, to allow
for a good comparability of results among different
centers (35-38).
69
Basso et al
TABLE I - POSSIBLE EXPLANATIONS FOR THE
DIFFERENCES REPORTED IN THE LIT-
ERATURE CONCERNING THE INCIDENCE
OF CD34
+
ACUTE LEUKEMIAS (AML AND
ALL)
1) Specimen analysed (bone marrow, peripheral blood)
2) Erythrocyte-lysed whole blood versus gradient density
mononuclear cell fractions
3) Use of cryopreserved versus fresh samples
4) Detection systems employed (flow cytometry, immuno-
fluorescence microscope, immunoenzymatic technique)
5) Use of different CD34 antibodies recognizing distinct CD34
epitopes (class I, II, III)
6) Degree of intensity for CD34 antigens
7) Cut-off levels for the discrimination of positive and negative
cases (5-20%)
8) Percentage of leukemic cells present in the sample
examined
9) Patients analysed (de novo AML or secondary AML;
childhood or adult ALL)
10) Biologic characteristics of acute leukemic cells (chromosome
aberrations, gene abnormalities)
11) Type of chemotherapy regimen employed

CD34 expression in acute leukemia
In recent years an accumulating number of recur-
rent genetic abnormalities have been reported in
AML, such as internal tandem duplication of Flt-3 re-
ceptor, ras-oncogene mutations, abnormalities of p53
tumor suppressor gene, p-glycoprotein expression, c-
myc amplification, hox overexpression or nm23 ex-
pression (1, 41). Although some of these genetic ab-
normalities are associated with specific AML FAB
subtypes, none of them represent causative lesions,
and with the exception of t(15; 17) in M3 AML they
may not be sufficient to induce the development of
malignancy, and therefore they probably represent
secondary hits in the multistep process of carcinogen-
esis (1, 44). There is also evidence that CD34
+
AML
are characterized by a higher incidence of abnormali-
ties involving chromosome 5 (-5; 5q-), 7 (-7; 7q-), 11
(11q23), t(9; 22), t(8; 21), and to a lesser extent chro-
mosome 16 (16q), 17 (17p), or complex karyotypes
(1, 5, 7, 9, 13, 18) (Tabs. II and III). Recent studies
have also found a close relationship between CD34
expression in AML and previous exposure to
chemotherapy, radiotherapy, some chemical com-
pounds, and/or pesticides (33, 34). CD34
+
AML are
also associated with trilineage myelodysplasia, as well
as dysgranulopoiesis (33) (Tab. II). In contrast, most a-
cute promyelocytic leukemias (M3) are CD34
-
.
Several papers have indicated that the immunophe-
notypic profile of CD34
+
AML is rather heterogeneous,
and this finding could explain some of the differences
reported in the literature regarding the biologic and
prognostic significance of CD34 expression in AML.
The antigenic profile of CD34
+
AML essentially de-
pends on the maturation stage and lineage commit-
ment of the leukemic clone. The correlation between
CD34
+
AML and FAB subtypes is illustrated in Table II.
Recent data have shown that biphenotypic acute
leukemias (BAL) are usually positive for CD34 antigen
(32, 42, 43). The large majority of CD34
+
AML co-ex-
press a number of antigens, which are not associated
with cell commitment, such as HLA-DR, CD38,
CD45RO, CD45RA, CD71, CD133 (AC133), CD123
70
TABLE II - CLINICO-BIOLOGICAL FEATURES OF
CD34
+
ACUTE MYELOID LEUKEMIAS
Incidence: 30-50% (de novo AML) ; 50-70% (secondary AML)
History: more frequently associated with previous exposure to
chemo-radiotherapy, pesticides or other leukemogenic
compounds (secondary AML)
Correlation with FAB subtypes: see Table III
Immunophenotypic profile: mainly dependent on the lineage
commitment and differentiation stage (frequent co-expression
of CD45, HLA-DR, CD38, CD33, CD13, CD133, CD117) (see
Tab. III)
Chromosome abnormalities: associated with the FAB
subtype and MIC correlates (the most frequently reported
abnormalities are the following: t(8;21);-5,-7,5q-,7q-, 11q23,
16q, 17p, complex karyotypes) (see Tab.III)
Cellular density for CD34: variable from case to case (range:
3.000- 130.000 per blast cell;higher in M2 t(8;21), M0 and M1)
Prognosis: variable from case to case, depending upon the
co-existence of specific genetic lesions
Therapy: chemotherapeutic regimens should be defined
according to accompanying genetic lesions
TABLE III - CD34 EXPRESSION IN DIFFERENT FAB SUBTYPES AND PRINCIPAL AML TRANSLOCATIONS
FAB CD34 Chromosome translocations CD34 Other
Subtype Expression and MIC correlates* Expression markers
M0 90-100% -5,-7,7q-,5q- 80-100%
t(9; 22) 70-90% KOR-SA3544
M1 70-90%
M2 20-40% t(8; 21) 50-90% CD19
(bright expression)
M3 0-5% t(15; 17) 0-3%
M3 variant 3-15% CD2
M4 20-60% M2/M4Eo/ inv(16) 30-70%
M2/M4Baso/ t(6; 9) 20-40%
M5a 50-80% t(9;11) 40-60% CD87(UPA-R)§
del11q(23)^ 50-70% 7.1
M5b 10-30%
M6 20-40%
M7 40-60% t(1;22) 50-60%
Biphenotypic
leukemia 90-100%
*MIC = Morphologic-Immunologic-Cytogenetic Classification of Acute Leukemias.
^ = See Reference 87.
§ = See Reference 88.

(IL-3 receptor alpha chain) (31, 32, 34, 37, 38, 44,
45). In contrast, only 10% of CD34
+
AML are CD90
positive. In addition, some surface and cytoplasmic
glycoproteins which are usually expressed by commit-
ted “myeloid” cells resulted positive in CD34
+
AML, i.e.
CD33, CD13, CD117 (stem cell factor receptor),
CDw116 (GM-CSF receptor), myeloperoxidase,
lysozyme (44-47). TdT (terminal deoxynucleotidyl
transferase) is restricted to M0-M1 and M4 subtypes,
while M6, M7, and M5b FAB subvarieties could ex-
press restricted lineage molecules, such as gly-
cophorin A, CD61 (glycoprotein IIIa), and CD14, re-
spectively (34, 44). Recently, the bright expression of
the P-170 glycoprotein on leukemic cells, which con-
fers resistance to several cytostatic drugs such as
antracyclines, vinca alkaloids, epipodophyllotoxins
and paclitaxel, has been correlated with CD34 expres-
sion, decreased response to treatment, and higher
levels of minimal residual disease in AML patients
(34, 37, 47). In some cases, an extended multiple
drug resistance (MDR) has been found in AML blasts
expressing high levels of P170 protein, suggesting
that these cells may be resistant also to other drugs
such as methotrexate, cisplatinum and alkylating
agents (48).
Raymakers et al (1995), have shown that the
CD34
+
/CD33
-
cell fraction obtained from the more dif-
ferentiated forms of AML (characterized by a few num-
ber of CD34
+
/CD33
-
cells) is exclusively constituted by
residual normal progenitors, and therefore distinct
from the leukemic clone, thus giving the possibility to
select this cell subpopulation for transplant purposes
(1995) (1, 50). Furthermore, it has been shown that
CD34 antigen expression on AML blasts having a
marked heterogeneity of cell size, is preferentially
found on small leukemic cells with rather low side
scatter; this feature was also found to be associated
with a shorter remission duration, and overall survival
rates, allowing the authors to speculate that this mor-
phological heterogeneity could reflect a peculiar bio-
logical behavior of AML (37). Recent data have shown
that the number of CD34
-
expressing cells increases
during evolution of myelodysplastic syndromes to
AML, and that the CD34
+
compartment develops a
growth advantage leading to a progressive expansion
of leukemic blasts during this period of time (51, 52).
Another source of variability in detecting CD34
+
AML is the type of CD34 monoclonal antibody utilized
for the immunophenotypic analysis. Numerous papers
have shown the existence of at least three distinct epi-
topes of the CD34 molecule, based on their differen-
tial sensitivity to the enzymatic cleavage (using neu-
raminidase, chymopapain, and glycoprotease), west-
ern-blot analysis, studies of cell reactivity, and cross-
blocking experiments (39, 40, 53-55). So far, more
than 30 different CD34 McAbs have been verified as
recognising the CD34 molecule, the most direct evi-
dence being reactivity with cells transfected with
CD34 cDNA and binding to CD34 glycoprotein (53).
Recent data have indicated that a number of AML
cases are positive for some CD34 McAbs and nega-
tive for others (especially if they belong to a different
epitope class), confirming the need to use the same
CD34 McAbs and the same detection technique in or-
der to achieve comparable results between different
centers (40, 53) (Fig. 1).
Another point which deserves careful discussion is
the level of expression for CD34 in AML. In normal
71
Basso et al
Fig. 1 - Normal maturation pathway of CD79
+
B-lymphocytes in the bone marrow according to CD10, CD34, and CD20.

CD34 expression in acute leukemia
hematopoiesis, CD34 antigen is expressed on virtual-
ly all the colony forming cells (CFU-C), and lympho-
cyte progenitors of either T or B lineage. However,
within the progenitor cell compartment, the degree of
positivity for CD34 decreases with cell differentiation
(maximum for multipotent cells and minimum for
unipotent cells), and disappears in more mature mor-
phologically identifiable bone marrow precursors (1,
31, 46, 47). Studies performed at the V and VI
International Workshop on Leukocyte Differentiation
Antigens (Boston, 1993; Osaka, 1996) allowed the
recognition of three main subsets of CD34
+
normal
bone marrow cells, having different CD34 antigen
density : low (2,000- 5,000 binding sites per cell-ABC),
medium (10,000-20,000 ABC), high intensities
(25,000- 40,000 ABC) (Fig. 2). This heterogeneity in
CD34 antigen expression in normal progenitors
makes it difficult to use this molecule alone for the
monitoring of minimal residual disease (MRD) in AML
patients treated with chemotherapy and/or bone mar-
row transplantation (37, 45, 47, 56). However, flow cy-
tometry quantification may allow the recognition of a
subgroup of CD34
+
AML, characterized by bright ex-
pression for CD34 (> 50,000 ABC), which could be
easily recognized even when present in a very low
number (< 0.1%) of nucleated cells. Leukemic cells
72
Fig. 2 - Expression of class I, II, and III CD34 McAbs in a patient with AML, M1 FAB subtype. A wide variation in cell reactivity was no-
ticed between the three McAbs used for the flow cytometry analysis.

overexpressing CD34 antigen may be detected in 10-
25% of the CD34
+
AML, while the remaining AML pa-
tients should be checked for their MRD by using alter-
native strategies such as the CD34/CD56;
CD34/CD65/TdT, CD34/CD14/HLA-DR; CD34/CD13/
CD33/ CD7 multiple stainings (37, 45, 56, 57). Lanza
et al have shown that CD34 overexpression could be
detected in M0-M2 FAB subtypes of AML (37, 40)
(Fig. 3). In another paper by MacDonald et al, CD34
overexpression was found to be strictly associated
with M2 FAB subtype of AML carrying t(8; 21) chro-
mosome alteration (24) (Tab.III).
CD34
AND ALL
The role of CD34 in acute lymphoblastic leukemias
(ALL) is more clearly defined than in AML although
significant changes have occurred over the last ten
years; in the early 90’s CD34 was considered a mark-
er of all precursor cells from the different lymphohe-
matopoietic lineages as well as a hallmark of acute
leukemias since these represent neoplasms of imma-
ture precursors.However, later studies showed the ex-
istence of CD34
-
ALL cases and the existence of a
negative association between this stem cell-associat-
ed antigen and positivity for other membrane B- and
T-cell differentiation markers such as CD20 or CD22
and CD3 (58). At present, it is clearly defined that
most (approximately 70 %) of common B (CD10
+
) ALL
cases (B-II) are CD34
+
, while a large percentage
(50%) of the phenotypically more immature pre pre B
ALL (CD10
-
) (B-I) results CD34-negative; in T-ALL
more than 40% of the cases results CD34 positive in-
dependently by the presence or absence of markers
of T-cell immaturity (30, 31, 59). Regarding the im-
munophenotypic expression of CD34 in normal BM
precursor B-lymphoid cells, this was definitively con-
firmed when Huang and Terstappen evidenced, in lim-
iting dilution experiments, the possibility that lymphoid
CD19 positive cells could derive from earliest CD34
+
(CD38
-
HLA-DR
+
) cells and that the co-expression of
CD19 and CD34 could be simultaneously detectable
in the earliest normal T- and B-cell committed precur-
sors (60). More recently, using multiparametric im-
munophenotyping, by three color analysis, different
groups have shown that the normal BM B-cell com-
partment is heterogeneous as far as the expression of
the various differentiation antigens is concerned; the
more immature B cells simultaneously express CD19,
CD10, and CD34, but lack CD20; later on, in the mat-
uration process CD34 and CD10 antigens sequential-
ly decrease in intensity and become negative. In par-
allel CD20 increases its intensity (Fig. 4).Today, CD34
is considered to be usually expressed in the early
phases of B-cell development and the presence of low
percentages of CD19
+
, CD10
+
and 34
+
cells in the BM
is considered a normal finding (58, 61-65).
The clinical significance of CD34 expression has
been analysed in several studies in both adult and
childhood ALL. Initial studies associated CD34 ex-
pression with a worse prognosis; however, this was
not confirmed by others which found no significant dif-
ferences in the outcome and the rate of complete re-
mission between CD34
+
and CD34
-
ALL cases.
Technical problems (Tab. IV) and particularly the dif-
ferent therapeutic approaches could contribute to ex-
plain at least to a certain extent these differences (59,
66-69).The hypothesis about the use of different ther-
apeutic regimens has been tested in correlating an
Italian series of pediatric patients obtained from
AIEOP (Italian Pediatric Association of Hematology
and Oncology) with two other studies in which an ade-
quate cohort of patients was studied. The first study
included 795 children older than 1 year from a
Pediatric Oncology Group (POG) in which for CD34
detection the My10 monoclonal antibody (class I ) was
used combined with an indirect immunofluorescence
technique. The percentage of 10% CD34
+
blast cells
was used as threshold for distinguishing positive from
negative precursor- ALLs. A correction factor was
used to exclude from the calculation of CD34
+
blast
cells contaminating the peripheral T lymphocytes.
CD34 expression was positively associated to low pe-
73
Basso et al
Fig. 3 - Comparative analysis of class I,II, and III CD34 epi-
topes-detecting McAbs by quantitative flow cytometry in nor-
mal BM cells, ALL and AML.
Fig. 4 - Quantitative analysis of CD34 antigen expression in
blasts obtained from different FAB subtypes of AML

CD34 expression in acute leukemia
ripheral WBC count (less than 50 x 10
9
/L) , no CNS
involvement, hyperdiploidy and absence of
cytogenetic translocations. The event free survival
was statistically worse in CD34
-
patients with respect
to those carrying this molecule and CD34 resulted as
an good independent prognostic marker in the multi-
variate analysis (58). Similar conclusions were ob-
tained later in the study from the St Jude’s Children’s
Research Hospital in a group of 335 children; in this
paper CD34 expression was assessed using the HP-
CA1 (class I) McAb and > 10% CD34
+
blast cells as a
threshold for CD34 positivity. Results confirmed that
the presence of CD34 was positively correlated in
common B-ALL (B II), with the most relevant favorable
prognostic factors including age 1 to 10 years, hyper-
ploidy, CD10 expression, no CNS disease; additional-
ly, absence of CD34 resulted in an independent ad-
verse prognostic factor in multivariate analysis. In this
study T-ALL was also analysed but no statistically sig-
nificant differences were found between CD34
+
(46%)
and CD34
-
ALL (59). The results of the two studies
could suggest that CD34 is an independent prognos-
tic factor in precursor B-ALL independent of the thera-
peutic regimen used to treat patients. In line with
these assumptions, in a retrospective study the ex-
pression of CD34 antigen in 708 children treated ac-
cording to intensive BFM oriented protocols (AIEOP
88’ and 91’) (69, 70), in which identical experimental
conditions to those of POG and St. Jude’s studies
(monoclonal antibody HPCA1, indirect immunofluo-
rescence technique, and the threshold) were used,
CD34 expression was also found to be associated
with a better clinical outcome.
Interestingly, the prednisone response (PR) evalua-
tion after 7 days did not significantly differ between
the CD34
+
(8.2%) and CD34
-
(6.1%) cases (p=0.8);
in a similar way morphologic complete remission rate
after one induction cycle was obtained in 97.3% of
CD34
+
and in 98.4% of CD34
-
ALL cases (p=0.8),
and relapse free survival was 74.2 + 2.8 versus 72.0
+ 5.7% (p=0.8). The conclusion of this study is that
CD34 expression is not clinically relevant once inten-
sive protocols such as the BFM oriented ones are
used, highlighting the considerations made above for
AML, that the therapeutic protocols are one of the
most relevant prognostic factors (33, 34). These re-
sults have been confirmed by several other studies in
which the undoable prognostic relevance of CD34 in
childhood ALL has not been found (59, 66-68).
Nevertheless it should be noted that, in contrast to
the potentially favorable clinical impact of CD34 ex-
pression in childhood ALL, in adults suffering from pre-
cursor B-ALL, CD34 has been frequently associated
with a worse prognosis and a poor outcome. Such dif-
ferences between childhood and adult ALL regarding
the clinical impact of CD34 expression could be relat-
ed to its associations with specific genetic markers of
good t(12; 21), hyperdiploidy, prognosis in children and
of bad prognosis t(9; 22) in adults as discussed below.
The multiparametric immunophenotyping based on
immunological gate in erythrocyte-lysed whole blood
was introduced in many laboratories in the late
nineties (30, 45, 61, 65, 69, 71, 72).The advantages of
this methodology are the correct identification and
quantification of the blast cells present in the samples
analysed even when they are present at low frequen-
cies (minimal residual disease) at the same time; this
facilitates its specific characterization. Assessment of
CD34 expression plays an important role in this con-
text since CD34 is expressed in a very high percent-
age of B-ALL (>75% once class III monoclonal anti-
bodies are used) while this antigen is expressed in a
very low percentage of cells in normal bone marrow
(1.5-2%), making co-expression of CD19 and CD34 a
powerful marker of ALL of B cell origin (60, 65, 72, 74).
Simultaneous detection of CD45 and CD34 is very
useful, in fact very few normal immature B lympho-
cytes express CD34 (less than 16 % of all CD19 in the
bone marrow) and CD45 shows a uniformly dim fluo-
rescence intensity, while in the majority of ALL CD45 is
either lower/ absent or much more heterogeneous (63,
65). Association with CD10 permits further distinction;
the expression of CD10
+
in the fraction CD34
+
is high-
er than in the fraction CD10
+
CD34
-
but frequently less
intense than in leukemic CD10
+
cells (63-65, 74).
These criteria are useful in solving relevant questions
in the management of treatment of ALL permitting a
good separation between normal regenerating B lym-
phocytes and possible recurrent ALL blasts.The CD34
associated with CD19 and several other markers such
as CD 45, CD10, CD38, TdT are, based on reported
reasons, good markers for the investigation of minimal
residual disease in ALL (45, 65, 73). Analogous results
could be obtained in the T-ALL using CD45, CD7,
CD34, and/or TdT and cyCD3 combination (59, 72).
Additionally, in recent years it has been shown that
74
TABLE IV - CD34 AND CD10 EXPRESSION IN THE MOST FREQUENT TRANSLOCATIONS ASSOCIATED WITH
PRIMITIVE B-ALL
Chromosomal translocation CD34 CD10 Prognostic impact
t(1;19) Negative (<700 MESF) Positive (100%) Good
t(12;21) Bimodal (96%) (5,100 MESF) Positive (100%) Excellent
t(4;11) Negative/Positive (50%) (4,800 MESF) Negative (100%) Poor
t(9;22) Unimodal (85%) (38,000 MESF) Positive (100%) Poor
Hyperdiploid Unimodal (100%) (8,340 MESF) Positive (100%) Excellent

75
Basso et al
CD34 plays a further relevant role in precursor B- ALL
in the identification of specific genotypes especially
when combined with CD10 and other markers.
Accordingly, translocation t(1; 19) (q23; p13) char-
acterized by E2A-PBX1 fusion protein identifies a sub-
type of pre B ALL (cµ
+
) in which CD34 is characteristi-
cally absent while CD10 is always present so this pat-
tern (CD34
-
CD10
+
cµ
+
) can be considered able to
identify this genetic abnormality (75, 76).
In a similar way, the t(12; 21)(p13; q22) transloca-
tion characterized by TEL-AML1 fusion protein is the
most frequent translocation in childhood B origin ALL
(25%) (72) and in this ALL sub-type, CD34 is always
positive with a bimodal expression in the context of
CD10
+
ALLs. In adult precursor B-ALL, t(9; 22) is as-
sociated with a high and homogeneous CD34 expres-
sion (77, 78, 80, 81). Using the multiparametric quan-
titative approach the sensitivity and specificity of the
immunophenotyping to identify these translocations is
>90% (81, 82).
In the t(4; 11) (q22; q23) translocation characterized
by ALL-MLL fusion gene, the CD10 is characteristical-
ly absent while CD34 is present in about 50% of
cases (82, 83).
Such differences in CD34 expression in the different
genotypic subtypes of precursor B-ALL help to explain
at least in part the controversial results frequently ob-
tained on the prognostic relevance of CD34 expres-
sion in precursor B- ALL; in fact in the CD34
+
group
the t(12; 21) and t(9; 22) ALL are included in children
and adults respectively, and they are characterized by
either an excellent prognosis t(12; 21) or a worse out-
come t(9; 22) (77,78); in the CD34
-
group t(1; 19) pre-
ALL is included, which is associated with a bad prog-
nosis and characterized by less intensive treatment
protocol (76, 84-86). As reported in Table IV the uti-
lization of CD34 in association with CD10 is powerful
in various genetic abnormalities identification, the fur-
ther association with CD45, CD20 and CD66c high-
lighting this ability (78-80).
CONCLUSIONS
Cellular expression of CD34 plays a relevant role
in acute leukemias and the CD34
+
represents a
heterogeneous group of leukemias characterized by
varying clinical, genetic and biological features; its
utility in defining specific disease subgroups re-
quires combined assessment with other immunolog-
ical markers. An effort towards the standardization
of methods, reagents, and therapy protocols to be
applied to de novo and secondary acute leukemias
should be made to achieve comparability of results
within and between different centers, as a basis for
quality assurance and quality control programs.
ACKNOWLEDGEMENTS
European Working Group on Clinical Cell Analysis
(EWGCCA) is supported in part by Concerted Action
Contract BMH4-97-2611 in the framework of the Biomed 2
programme (European Commission, Brussells, Belgium).
This work was also supported by AIRC, CNR (Progetto
Strategico “Oncologia”), MURST 40 e 60%, e Fondazione
Città della Speranza di Padova.
Reprint requests to:
Prof.Giuseppe Basso
Dipartimento di Pediatria
Università di Padova
Via Giustiniani, 3
35128 Padova
gbasso@oncopedipd.org
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